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By Dennis Miller, BHC Senior Writer

A new study provides important clues about the mechanism behind cocaine addiction, and may offer new avenues for pharmacologic treatment. The study, published in the journal Science, reveals genetic changes which can occur in the brain as a result of repeated exposure to cocaine. The study’s results join a growing body of research in the relatively new branch of science called epigenetics, which explores how lifestyle and environmental effects may change gene expression over time.

The research team was lead by Ian Maze, a Ph.D. student in the Department of Neuroscience at the Mt. Sinai School of Medicine. “We identified a gene known as G9a whose job is basically to maintain homeostatic levels of the gene expression in cells,” Maze explains. “Following a single dose of cocaine, you have increased expression of this particular gene and protein, G9a. And once it’s increased its expression, it’s very rapidly recruited to turn genes off that become inappropriately expressed or turned on in response to that single dose of cocaine.”

That response to a single dose of cocaine dose is what the G9a protein is supposed to do — regulate and mitigate the brain’s response to new chemical stimuli to ensure that genes are not switched on or off aberrantly. “The normal function of G9a is to basically maintain normal levels of gene expression” says Maze. “And so, it very effectively and efficiently does its job in response to its first dose of the drug.”

But over time, as cocaine exposure is repeated, the protein’s reaction appears to lessen, almost as if the brain begins to accept the presence of cocaine as normal. “Following repeated dosing of the drug, G9a expression is actually reduced and its activity is reduced,” Maze explains. “Therefore, a number of genes that turn on in an aberrant fashion in response to repeated cocaine are now unable to be returned to their normal basal level of gene expression. A lot of these genes that are turning on inappropriately are involved in synaptic plasticity, which means that you’re actually having new synaptic expressions forming in response to repeated cocaine, as opposed to acute, and this leads to increased behavioral sensitivity to the drug.”

The study was funded by the National Institute of Drug Abuse (NIDA). In a statement, NIDA Director Nora Volkow said the finding is potentially groundbreaking. “This fundamental discovery advances our understanding of how cocaine addiction works. Although more research will be required, these findings have identified a key new player in the molecular cascade triggered by repeated cocaine exposure, and thus a potential novel target for the development of addiction medications.”

The study’s findings build on previous research which had already shown that cocaine can induce changes in gene expression. This research deepens the understanding of the precise biochemical mechanism through which that occurs. “We have known before that repeated cocaine use leads to increased gene expression,” says Maze, “However, nobody had actually ever shown a role for histone methylation, which G9a basically controls. Nobody had ever really provided a solid mechanism that may help to explain some of the persistent changes in gene expression that we see.”

The potential for the discovery to lead to more effective treatments for addiction to cocaine (and potentially for other drugs as well) lies in this more precise identification of the specific mechanism behind the addictive response. “The importance of this study is that it explains a novel mechanism by which synaptic plasticity occurs following exposure to drugs of abuse,” Maze explains. “And it helps us to identify new genes that are being regulated by this molecule that may actually end up being good targets for future therapeutics.”

In fact, in the study, Maze and his team were actually able to demonstrate that by countering the effect of repeated cocaine exposure’s on G9a, they could blunt cocaine’s addictive effect. “If cocaine’s job is to reduce G9a, then G9a being reduced would lead to this increase in sensitivity to the drug,” Maze says. “And of course, we found the opposite — when you oppose the effects of cocaine and you over-express G9a, you can completely block increased sensitivity to the drug.”

To listen to our full interview with Ian Maze, click on the audio icon above. The following is an edited written transcript.

BHC: Give us an overview of the study and what it found.

IM: Basically, what we were interested in is we were trying to identify potential modifications that may help to explain some of the persistent behavioral effects of cocaine use. And so in doing so we identified a gene known as G9a whose job is basically to maintain homeostatic levels of the gene expression in cells — and it does this of course in normal cells as well.

And so basically what we found is that following a single dose of cocaine, you have increased expression of this particular gene and protein, G9a. And once it’s increased its expression, it’s very rapidly recruited to turn genes off that become inappropriately expressed or turned on in response to that single dose of cocaine.

However, what we found is that following repeated dosing of the drug, G9a expression is actually reduced and its activity is reduced. Therefore, a number of genes that turn on in an aberrant fashion in response to repeated cocaine are now unable to be returned to their normal basal level of gene expression. And therefore, a lot of these genes that are turning on inappropriately are involved in synaptic plasticity, which basically means that you’re actually having new synaptic expressions forming in response to repeated cocaine, as opposed to acute, and this leads to increased behavioral sensitivity to the drug.

BHC: Any idea why it takes prolonged exposure to produce this change and why a single exposure doesn’t seem to do it?

IM: Yes. Basically, I think that the normal function, as I mentioned, of G9a is to basically maintain normal levels of gene expression. And so, it very effectively and efficiently does its job in response to its first dose of the drug.

However, as you start to take drugs repeatedly, other signaling cascades in the brain become activated and one of them leads to the production of a protein known as ∆FosB [pronounced delta FosB]. ∆ FosB has been shown to play a very important role in the addictive-like process, as this protein actually accumulates throughout the reward circuitry following repeated exposure to drugs of abuse — multiple drugs of abuse, including cocaine.

Now, as ∆ FosB accumulates in the brain, it actually feeds back onto G9a to basically repress G9a expression. And in doing so, then, as I mentioned, all these genes start to turn on inappropriately that G9a usually represses.

BHC: How much of this was known before or perhaps theorized before?

IM: We have known before that repeated cocaine use leads to increased gene expression and people, for years, have taken candidate approaches to try to study how those individual genes may be involved in the development of the addictive behaviors that we see in rodent models. However, nobody had actually ever shown a role for histone methylation, which basically G9a controls.

So basically, yes, people had shown before that gene expression is increased in response to repeated cocaine, but nobody had ever really provided a solid mechanism that may help to explain some of the persistent changes in gene expression that we see. And so, G9a acts as a kind of a novel molecule and mechanism to control these aberrant changes in G9a that we see.

BHC: Tell us a little bit more about the science of epigenetics, which I understand this study is on the leading edge of.

IM: I’ll put it in the context of addiction. I think that, obviously, there is a very solid genetic component to drug addiction itself, in that people have done a number of studies throughout the years looking at family histories of drug abuse, and then showing that individuals who have, say, a family history of drug abuse tend to be more susceptible or predisposed to becoming addicts themselves. The problem with these studies is that although there is a strong genetic component, it only helps to explain a small percentage of actual drug addicts. Many drug addicts start taking drugs chronically throughout their lifetimes and become addicted, and they have no family history for that drug abuse.

So, there appears to be some kind of a gene/environment interaction going on there — it’s not just at the level of genetics itself. And so, people theorized that potentially what’s happening in this is that it’s not exactly a genetic mutation that’s leading to the drug abuse itself, but that the ways our genes are expressed in the brain may actually be contributing to this process. And therefore, what epigenetics describes is not — it’s not the DNA itself but how the DNA is expressed in different cells.

And so, just to put it in perspective, every cell in our body — whether it be a cardiac cell, neuronal cell, a liver cell — have the same genetic code. What makes the cells unique is how those genes that are expressed in those cells are actually turned on or turned off — the patterns in which they’re turned on and turned off.

And so, what we’re talking about in terms of epigenetics is how repeated use of cocaine is actually altering the ways in which genes are being turned on and turned off in these neural cells within the reward circuitry of the brain.

BHC: Ian, can you hypothesize of ways that this finding might lead to some better treatments for addiction?

IM: Sure. I think that in terms of development of novel therapeutics for the future, I think that these findings kind of open up a new area for gene discovery. So I don’t believe that necessarily targeting G9a is going to lead to a novel therapeutic approach, because G9a is kind of a master gene regulator, so if you alter G9a’s function throughout the body and systemically, chances are it’s going to have negative and adverse side effects.

However, what the importance of this study is, is that it explains a novel mechanism to which synaptic plasticity occurs following exposure to drugs of abuse and it helps us to identify new genes that are being regulated by this molecule that may actually end up being good targets for future therapeutics.

BHC: Ian, is your finding limited strictly to cocaine addiction or do you suspect that the finding may be just as true for other substances or perhaps even process addictions such as to gambling or to sex?

IM: I believe it’s probably not limited just to cocaine. Given that G9a is regulated so heavily by this molecule ∆ FosB that I mentioned earlier. ∆ FosB is a protein that accumulates in response to numerous different drugs of abuse, including cocaine, heroine, morphine, alcohol, etc. And since these drugs induce ∆ FosB, I think it’s reasonable to hypothesize that ∆ FosB will have a similar effect on G9a expression following exposure to other drugs of abuse.

I can tell you, although the data is preliminary and unpublished as of yet, it does appear that G9a’s role in the regulation of behavior in response to other drugs, including morphine, appear to be very similar to what we see with repeated cocaine. So I think that G9a is probably very important to the addiction process as a whole and not just to cocaine addiction itself.

BHC: Give us a quick overview of the methodology of the study.

IM: I think it takes on a number of different components. When we began the study, we were trying to identify which of these chromatin modifiers, or these epigenetic modifiers may be regulated by repeated cocaine. So in order to do this, we actually began by profiling the messenger RNA expression of these different important modifiers, trying to identify if any of them showed altered transcript expression following repeated cocaine.

After we identified that G9a and its binding partner the G9a-like protein, or GLP, were both significantly reduced following repeated cocaine, we of course verified these findings using protein biochemistry to verify that the proteins themselves were being disrupted.

Following these types of studies, we also were able to then manipulate G9a specifically within the nucleus accumbens (which is the region of the rewards circuitry that we’re studying) by basically cloning G9a into a virus and being able to inject that virus specifically into these neuronal populations in the reward circuitry, and then either over-express or knock G9a out, and then monitor how these animals responded to cocaine. And basically we were able to show that if we mimic the effects of repeated cocaine, which is reducing G9a expression, then we actually enhance behavioral phenotypes and behavioral sensitivity to cocaine.

This started to make sense in that, if cocaine’s job is to reduce G9a, then G9a being reduced would lead to this increase in sensitivity to the drug. And of course, we found the opposite — when you oppose the effects of cocaine and you over-express G9a, you can completely block increased sensitivity to the drug.

Finally, in terms of trying to identify the actual mechanism through how this is controlled, we used a number of different techniques including something called chromatin immunoprecipitation, which allows us to actually identify where G9a is binding throughout the genome and which genes it’s directly regulating following repeated cocaine, and that allowed me to identify a number of genes that are involved in forming new synaptic connections as potential targets of G9a. And these types of targets are what we would probably go after in the future for potential therapeutics.

BHC: Well, excellent, Ian. Anything we haven’t touched on here that you would like to mention in closing?

IM: I guess I can go ahead and just summarize. And I would just say that, I think in summary, G9a normally functions within the normal nervous system reward circuitry to block the ability for environmental perturbations to aberrantly induce gene expression. I think though, however, following chronic cocaine or repeated exposure to cocaine, G9a activity appears to be reduced, which leads to the activation of many genes that contribute to enhance behavioral sensitivity and heighten synaptic connectivity between neurons, thus strengthening associations between the drug experience itself and the neuron reward circuitry potentially leading to addiction.

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